Development and Use of Cellular Systems to Assess and Correct Splicing Defects

Abstract

A significant proportion of mutations underlying genetic disorders affect pre-mRNA splicing, generally causing partial or total skipping of exons, and/or inclusion of pseudoexons. These changes often lead to the formation of aberrant transcripts that can induce nonsense-mediated decay, and a subsequent lack of functional protein. For some genetic disorders, including inherited retinal diseases (IRDs), reproducing splicing dynamics in vitro is a challenge due to the specific environment provided by, e.g. the retinal tissue, cells of which cannot be easily obtained and/or cultured. Here, we describe how to engineer splicing vectors, validate the reliability and reproducibility of alternative cellular systems, assess pre-mRNA splicing defects involved in IRD, and finally correct those by using antisense oligonucleotide-based strategies.

Keywords: ABCA4; Antisense oligonucleotide; Exon skipping; Genetic therapy; Inherited retinal diseases; Maxigene; Midigene; Pre-mRNA; Pseudoexon; Splicing modulation; Splicing vectors.